This invention relates to a method of assaying a bioactive component in a biological specimen by an assay with the use of an antibody, a binding protein or a receptor.
Many bioactive components such as growth factors, hormones, vitamins and medicaments are bound to binding proteins so that the effects and metabolic speeds thereof are controlled in vivo. In case of assaying bioactive components by assay with the use of antibodies, binding proteins or receptors, these coexisting binding proteins frequently affect the assay data.
In case where a substance to be assayed coexists with binding proteins in a biological specimen, it has been a practice to carry out various pretreatment to eliminate the effects of the binding proteins on the assay. In the case of insulin-like growth factor 1 (IGF-I), for example, the xe2x80x9cacid-ethanol methodxe2x80x9d may be cited as the method which is most commonly employed at present (W. H. Daughaday, Journal of Clinical Endocrinology and Metabolism, 1980, Vol. 51, p. 781-788). This method utilizes a phenomenon that when a biological specimen is treated with a mixture of hydrochloric acid with ethanol, binding proteins releasing IGF-I under acidic conditions become insoluble in the ethanol atmosphere and thus can be easily eliminated from the specimen by centrifugation.
In the case where centrifugation is omitted in the acid- ethanol method, however, the binding activity of the binding proteins to IGF-I is restored at the point of neutralization and thus affects the assay data. To overcome this problem, JP-A-8-145998 reports a method wherein a re-binding inhibitor is added to a neutralizing buffer to be used after the acid treatment (the term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined published Japanese patent applicationxe2x80x9d). Since it is considered that IGF-I all exists in the free form in the neutralized biological specimen solution in this method, the assay data are not affected by the coexisting binding proteins. As the re-binding inhibitor, use is made of 8-anilino-1-naphthalenesulfonic acid salt (ANS) and the like.
ANS, which has been used for a long time to release thyroid hormone from binding proteins, suffers from some problems such as being unstable to light and atmospheric oxidation and having a strong toxicity. In addition, it is pointed out that ANS sometimes affects immuno reactions, which restricts the utilization of ANS in assaying biological specimens.
As methods for eliminating the effects of binding proteins contained in specimens, Japanese Patent No. 1,940,596 proposes a method wherein, in analyzing vitamin B12, thiol group is introduced into binding proteins so as to inactivate the binding activity to vitamin B12 while Japanese Patent No. 2,023,927 proposes a method wherein binding proteins are denatured by using a peroxy acid. However, denaturing agents should be added in excess in these known methods and it is therefore needed to inactivate the excessive denaturing agents after the completion of the denaturation of the binding proteins.
There is known a method of assaying IGF-I by adding IGF-II in excess to a specimen. Since IGF-II blocks all of the binding sites of binding proteins, IGF-I, which has been thus driven away and released, is immunologically assayed in this method (W. F. Blum, Acta Endocrinokogica, 1988, Vol. 118, p.374-380). In this method, it is necessary to use antibodies which would not have crossreactivity with IGF-II. Since IGF-I is closely similar in structure to IGF-II, it is highly difficult to obtain antibodies which are never affected by IGF-II added in excess in the pretreatment. Although JP-A-6-102275 reports a similar method for assaying a steroid hormone, this method suffers from some problems such that an assay system usable therefor is restricted and costs a great deal.
To eliminate the effects of binding proteins coexisting in biological specimens, it is needed in the conventional art to add expensive reagents or highly toxic reagents to pretreating agents, neutralizing agents or assay buffers or to employ troublesome procedures or special instruments, as described above. Therefore, it has been required to solve these problems encountering in the existing methods.
It has been found out in the invention that when a specific pretreating agent is added to a specimen at such a level as not affecting the activity of the substance to be assayed, binding proteins are exclusively inactivated and the thus obtained mixture can be subjected to the assay as such.
That is to say, by exposing a biological specimen to a surfactant and/or an alkali agent, the binding proteins are released from a substance to be assayed and irreversibly denatured simultaneously. The thus pretreated specimen may be neutralized or diluted so that the pH value attains a level appropriate for the assay, while requiring neither neutralization of a functional group of a denaturing agent nor addition of any special material preventing the substance to be assayed from re-binding to the binding proteins. Alternatively, the pretreated specimen may be subjected to the assay as such.